PROJECT SUMMARY/ABSTRACT ! This project aims at elucidating previously unknown aspects of termination factor Rho action in bacterial cell. We have discovered that Rho is a part of much larger than expected regulatory network that includes riboswitches and sRNAs. We also discovered a number of protein-protein interactions formed by Rho, independent of nucleic acids, allowing its effects on global gene expression to be modulated by the upstream regulatory signals. In this proposal we build upon these discoveries by deploying multi-faceted yet integrated analytical tools focusing on Rho interactions and non-canonical functions in the cell. First, we will use in vivo structural interactomics to discover protein complexes formed by Rho, reconstitute them in vitro and subject them to an array of complimentary methods of structural interrogation (electron microscopy, covalent cross-link mapping, and chemical footprinting). Protein-protein interactions thus discovered will then be targeted by structure-based designed mutations and peptidomimetics. These in turn will allow to study individual aspects of Rho action as a global regulator of gene expression without affecting its other functions. Second, we are going to expand our research of RNA-mediated regulation of Rho function in the cell by including new targets and new classes of RNA (e.g. non-coding RNAs). By deploying next generation sequences approaches we will generate a comprehensive network map of these previously overlooked regulatory mechanisms. Third, in addition to structural characterization of Rho-nucleated protein-protein complexes, we will characterize in detail the effects Rho-interactors have on its function in vivo and in vivo, and elucidate the mechanism of each effect. Our preliminary data indicates that a variety of mechanisms can be employed by these (often uncharacterized) regulators, from simple competitive inhibition of Rho association with RNA polymerase to its covalent modification(s). The impact of the proposed research will be as multi-faceted as its approach. It will provide the first comprehensive and detailed picture of Rho as a global regulator of gene expression in bacteria. The field of transcription regulation will be provided with solid structural framework to assist in interpreting existing data and directing future studies, as well as the novel tools to be deployed therein. Given Rho impact of expression of horizontally transferred genes, including pathogenicity islands, this research will have a positive impact on understanding of regulation of bacterial virulence, whereas peptidomimetics disrupting Rho function can serve as novel lead compounds in development of anti-bacterials.